To date, mainly two recombination pathways, namely, a homologous recombination pathway and a non-homologous recombination pathway have been identified in eukaryotic cells. Homologous recombination is induced by the interaction between homologous sequences of DNA, whereas non-homologous recombination is irrelevant to such DNA homology and it is considered to conduct a direct ligation of cleaved double-stranded ends. In the case of budding yeast, a homologous recombination system has mainly been used as a recombination mechanism. If foreign DNA has a portion homologous to the genomic sequence of DNA, into which it is to be incorporated, at both ends thereof, the foreign DNA can be incorporated into the genomic site homologous to the sequence (Takata et al., 1997; Wach et al., 1994). It has been reported that Rad51, Rad52, and Rad54 are essential in this process (Nickoloff and Hoekstra, 1998). On the other hand, many other living bodies including humans, plants, insects, and fission yeasts have mainly used a non-homologous recombination system as a recombination function. In these living bodies, even if foreign DNA has a long DNA sequence portion that is homologous to a specific region on the genome, it is incorporated into the specific region with low frequency, and it is incorporated at random into the genome in many cases.
Homologous recombination enables efficient modification of the existing genes. Since it can be used for the production of a new species of strains or the improvement of decreased functions of cells, a large number of attempts to increase the ratio of homologous recombination have been made in eukaryotic cells other than budding yeasts, to date.
For example, an attempt to construct a high expression system of the RAD51 gene, RAD52 gene, or the homolog gene thereof, which plays an important role in the homologous recombination of budding yeasts, has been made. However, it has been known that even if such RAD51 or RAD52 is allowed to express at a high level, homologous recombination ratio is increased only by approximately 2 or 3 times, and that it rather adversely affects cells (Yanez and Porter, 2002; Reiss et al., 2000). In addition, various types of targeting vectors have been developed to increase the ratio of homologous recombination. For example, a method to concentrate homologous recombinants (please refer to patent Document 1 and Non-Patent Documents 1 and 2) based on the negative-positive selective method in mammalian cells or plant cells is a representative example. However, even if such a method is applied, homologous recombination frequency is still extremely low (1% or less). Moreover, since application of such a method requires complicated operations, this has not been a practical method.
With regard to studies about genetic recombination in eukaryotic cells other than budding yeasts, since genetic approach can easily be carried out, such studies have been conducted not only using fission yeasts but also using filamentous fungi. A type of filamentous fungi, Neurospora crassa, is one of organisms often used in studies regarding recombination. It has been known that the mei-3, mus-11, and mus-25 genes of Neurospora crassa are homologous to RAD51, RAD52, and RAD54, respectively, which function in the homologous recombination of budding yeasts. Thus, the ratio of homologous recombination of a mutant comprising a deletion regarding these genes has been studied (Handa et al., 2000) by measurement of homologous-integration frequency of the mtr gene contained in the plasmid pMTR (Schroeder et al., 1995) into the chromosomal mtr locus as an indicator. Only 3% to 5% of transformants exhibited homologous integration in wild-type strain. In contrast, in the case of mei-3 and mus-25 mutants, almost no such homologous recombination took place. These data also showed that the ratio of homologous recombination is extremely low in Neurospora crassa, and that it is not easy to disrupt a specific gene by gene targeting.
On the other hand, it has been reported that a non-homologous recombination process progresses via DNA-dependent protein kinase (DNA-PKcs), a K70-Ku80 heterodimer, and a DNA ligase IV-Xrcc4 complex (please refer to Non-Patent Documents 3, 4, and 5). Thus, the inventor has conducted studies based on a working hypothesis that the ratio of homologous recombination would be increased by inhibition of the non-homologous mechanism.    Patent Document 1: Japanese Patent Application Laid-Open No. 2001-046053    Non-Patent Document 1: Terada et al., Nature biotech. 20, 1030-1034. 2002    Non-Patent Document 2: Jeannotte et al., E J. Mol. Cell Biol. 11, 5578-5585. 1991    Non-Patent Document 3: Gallego et al., the Plant Journal, 35, 557-565. 2003    Non-Patent Document 4: Walker et al., Nature 412, 607-614. 2001    Non-Patent Document 5: Critchlow and Jackson, TIBS, 23, 394-398. 1998